Development of a loop-mediated isothermal amplification (LAMP) assay based on the C962R gene for african swine fever virus detection
Abstract
Aim. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for African swine fever virus (ASFV) detection. Methods. Primer design was performed using publicly available full genome sequences of ASFV. A panel of heterologous DNA samples and reference ASFV DNA samples were used for the assay specificity testing. The limit of detection (LOD) was assessed using purified and quantified serial dilution of the amplified target sequence. LAMP product detection was performed via gel-electrophoresis and via ethidium bromide fluorescence under UV after adding the ethidium bromide directly to the tube with the LAMP product. Results. Three primer sets amplifying different regions of ASFV gene C962R were developed, of which the set № 2 providing the most intense product synthesis with the most vivid and clear pattern was selected for further studies. The optimal concentration of reaction mix components for the most effective primer set was established. In the final protocol the LAMP reaction was carried out at 60 °C for 40 min. The limit of detection (LOD) of the assay was 50 copies of the target sequence per reaction. In a preliminary testing the assay proved specific, using 10 reference and 4 heterologous viral and two bacterial DNA samples. Our LAMP assay detected ASFV genotypes I and II that are currently spread in Europe, Asia, and the Pacific and IX, occurring in Africa. Conclusion. A LAMP assay was developed based on the C962R gene that proved in preliminary validation to be specific and sensitive and was able to detect down to 50 copies per reaction of purified target gene within 40 minutes. Classical gel electrophoresis and direct staining using ethidium bromide were used for product visualisation in this study. Colorimetric approaches or the use of lateral flow devices in the visuali- sation step could make the assay less equipment dependent. Further validation of the assay, determining analytical specificity, selectivity and reproducibility performance characteristics also using clinical samples under field condi- tions and inclusion of an internal control would possibly enable its use as a test of choice at point-of-care and at low resource laboratories.References
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